The hypothesis that will be tested in this proposal is that specific signaling pathways are involved in the epithelial-mesenchymal interactions, between hair follicle stem cells and the specialized mesenchymal cells of the dermal papilla, during hair follicle regeneration. The hair follicle is an ideal model for studying epithelial-mesenchymal interactions, and a better understanding of the extracellular and intracellular signaling pathways involved in hair follicle stem cell proliferation and differentiation may lead to advances in the areas of tumorigenesis, wound healing, aging, alopecia and hair disorders. We previously identified keratin 15 (K15) as a marker of hair follicle stem cells. We demonstrated that K15 expression defines the human hair follicle bulge area and these K15+ cells possess the biologic properties of stem cells by several criteria. In telogen follicles, K15 is expressed in the basal layer of the club hair structure, which can be removed by plucking from dispase treated scalp skin. Since the lower follicle degenerates in the catagen phase, leaving only the telogen club structure, this residual epithelial club hair represents an enriched source of K15+ stem cells which are responsible for regenerating a new lower follicle upon stimulation by the dermal papilla. In addition, the dermal papilla and fibrous connective sheath of the hair follicle bulb have been shown to possess the inductive capacity to form hair follicles from the epidermis. Using co-culturing techniques, cDNA expression arrays and other gene expression analyses, I will study the signaling pathways involved in the epithelial-mesenchymal interactions between human hair follicle stem cells and dermal papilla cells: Aim 1. To characterize gene expression during early hair, follicle induction, between the hair follicle keratinocyte stem cells and the dermal papilla cells, in an in vitro model system. Hypothesis: Specific extracellular and intracellular pathways are involved in signaling between the keratinocytes and dermal papilla cells. Cultured human hair follicle keratinocyte stem cells and dermal papilla cells will be combined in a co-culturing system where the cells are separated by a thin membrane which allows for the free flow of media and proteins. At several defined time points, the keratinocytes and dermal papilla cells will be harvested and total RNA will be extracted. cDNA expression array analysis will be performed to study the pattern of gene expression during this in vitro model of early hair follicle regenerative induction. Aim 2. To verify and further characterize the expression of cell signaling genes of the keratinocyte stem cells and dermal papilla cells within tissue samples. Hypothesis: Signaling genes identified by in vitro methods are involved in the induction of hair follicle regeneration at the onset of anagen in vivo. The expression of signaling genes identified by cDNA array analysis will be confirmed by rt-PCR on human scalp samples. Positive transcripts will be further investigated for upregulated expression within the hair follicle at the onset of anagen. Frozen section slides of scalp will be subjected to laser-capture microdissection to obtain RNA from the stem cell-rich bulge area of follicles in full anagen, when the stem cells are quiescent, and at the onset of anagen, when the stem cells are induced to proliferate and differentiate. The samples will be analyzed by real-time PCR to compare the relative levels of gene expression. Dermal papilla cell gene expression will be similarly studied.